J Mol Med DOI 10.1007/s00109-013-1048-7 REVIEW Retrograde trafficking of AB5 toxins: mechanisms to therapeutics Somshuvra Mukhopadhyay & Adam D. Linstedt Received: 1 April 2013 /Revised: 23 April 2013 /Accepted: 24 April 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract Bacterial AB5 toxins are a clinically relevant class and epidemic diarrhea; and pertussis toxin (PTx), which is the of exotoxins that include several well-known members such causative agent for whooping cough [1, 2]. Each year, infec- as Shiga, cholera, and pertussis toxins. Infections with toxin- tions with these toxin-producing bacteria affect millions of producing bacteria cause devastating human diseases that individuals and cause more than a million deaths [1]. affect millions of individuals each year and have no definitive AB5 toxins are so-called because they are formed by the medical treatment. The molecular targets of AB5 toxins reside association of a single A subunit with a pentameric B subunit in the cytosol of infected cells, and the toxins reach the cytosol (Fig. 1)[1, 2]. The toxins exert their cytotoxic effect by by trafficking through the retrograde membrane transport altering the activity of specific molecular targets in the cytosol pathway that avoids degradative late endosomes and lyso- of infected cells. STx blocks protein synthesis by removing a somes. Focusing on Shiga toxin as the archetype member, single adenine residue from the 28S ribosomal RNA, and CTx we review recent advances in understanding the molecular and PTx increase cAMP levels by ADP-ribosylating the Gsα mechanisms involved in the retrograde trafficking of AB5 or Giα components of heterotrimeric G proteins, respectively toxins and highlight how these basic science advances are [1, 2]. While the catalytic activity of the toxins resides in the A leading to the development of a promising new therapeutic subunit, retrograde trafficking is mediated by the pentameric approach based on inhibiting toxin transport. Bsubunits[1]. As retrograde trafficking is a prerequisite for productive infections, there is significant interest in designing Keywords Retrograde trafficking . AB5 toxins . small molecule inhibitors of B subunit trafficking that may be Mechanisms . Therapeutics therapeutically useful [3–5]. Over the last two decades, the transport of STx has been more extensively studied than other AB5 toxins. Conse- Introduction quently, our understanding of STx transport is more ad- vanced than that of other toxins. However, studies AB5 toxins are a biomedically important class of bacterial performed on other AB5 toxins suggest that there are com- exotoxins that cause devastating human diseases. Prominent mon thematic and conceptual similarities in toxin transport members of this class include Shiga toxin (STx), which causes although specific molecular factors may differ. Here, we life-threatening diarrhea, dysentery, hemorrhagic colitis, and review the important steps in the retrograde trafficking of hemorrhagic uremic syndrome; cholera toxin (CTx) and AB5 toxins with a specific focus on STx transport and Escherichia coli heat-labile enterotoxins, which cause endemic summarize recent progress in developing small molecule inhibitors of toxin trafficking. S. Mukhopadhyay Division of Pharmacology & Toxicology, College of Pharmacy and Institute for Cellular & Molecular Biology, The University of Texas at Austin, Retrograde trafficking to the endoplasmic reticulum Austin, TX 78712, USA STx and other AB toxins follow an elaborate retrograde * 5 A. D. Linstedt ( ) trafficking pathway to reach the cytosol from the cell exterior. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA Trafficking, mediated by the B subunits, begins at the plasma e-mail: [email protected] membrane where the toxins bind cell surface receptors. After J Mol Med Fig. 1 Subunit structure of AB5 toxins. Shiga toxin (STx, PDB ID: 1R4Q [88]) and cholera toxin (CTx, PDB ID: 1XTC [89]) are compared as representative members of the AB5 toxin family. Each subunit is distinctly colored. Catalytic A subunits have a single alpha helix penetrating the center of the pentameric B subunits. The basal surface of the B subunit pentamer contains binding sites for the glycolipid receptors and is shown in the lower panel internalization, the toxins sequentially traffic through sorting Endocytosis The glycolipid globotriaosylceramide (GB3) endosomes and the Golgi apparatus to reach the endoplasmic acts as the functional cell surface receptor for STx reticulum from where the A subunit is translocated to the [14–16]. Receptor binding is essential for endocytosis. Mice cytosol (Fig. 2)[6–11]. Studies on toxin trafficking have depleted in the GB3 synthase gene are resistant to Shiga contributed immensely to our understanding of retrograde toxicosis [17], and in humans, endothelial cells of the micro- transport in general. Work on STx demonstrated, for the first vasculature particularly those in the kidney are primary targets time, that exogenous proteins internalized by endocytosis can of STx because they express high levels of GB3 [18, 19]. be transported to the Golgi apparatus and the endoplasmic reticulum [12]. Later studies on STx led to the discovery of the Clathrin-dependent endocytosis Studies show that both direct transport pathway between sorting endosomes and the clathrin-dependent and clathrin-independent processes are in- Golgi apparatus that bypasses degradative late endosomes volved in STx endocytosis (Fig. 3). Ultrastructural analyses [13]. Numerous endogenous proteins are now known to traffic performed on HeLa cells incubated with STx reveal that at low via the retrograde pathway suggesting that the toxins co-opt a temperatures (0 °C), STx diffusely binds the plasma mem- preexisting endogenous pathway. brane, but a short incubation at 37 °C leads to accumulation Fig. 2 Retrograde trafficking pathway of STx and other AB5 toxins. Toxins bind the cell surface, undergo endocytosis, and enter sorting endosomes where they move into Golgi- directed tubular extensions. Trafficking to the Golgi bypasses degradative late endosomes (LE) and lysosomes. From the Golgi, the toxins move to the endoplasmic reticulum, and then, the A subunits are transported to the cytoplasm where they affect specific molecular targets J Mol Med Fig. 3 Clathrin-dependent and clathrin-independent endocytosis of STx. The schematic diagram shows speculative relationship of two of the pathways by which STx gains access to the cell interior. Components known to be required in the respective pathways are highlighted. Also indicated are the upregulation of the clathrin-mediated pathway by STx-induced syk signaling and the ability of STx itself to induce membrane curvature leading to non- clathrin-mediated internalization of STx in coated pits, which are sites of clathrin-mediated category [25]. Further, the actin-based endocytic processes of endocytosis, as well as in uncoated pits [20]. When clathrin- macropinocytosis and phagocytosis are also clathrin indepen- mediated endocytosis is blocked by depleting clathrin heavy dent and not included in the above classification [25]. The role chain using small interfering RNA (siRNA), endocytosis of the of individual clathrin-independent pathways in STx endocyto- B subunit of STx (STxB, which shows similar transport sis has not yet been rigorously investigated but, at the mini- kinetics to the STx holotoxin), decreases by ∼40 % while the mum, there is clear evidence that dynamin-independent same conditions reduce endocytosis of the clathrin pathway internalization can take place. One study reported that inhibi- marker transferrin by ∼80 % [21]. Moreover, expression of tion of dynamin activity by expression of a dominant negative dominant negative mutants of epsin or eps15 (proteins required mutant of dynamin (K44A) reduces STx endocytosis by only for clathrin-mediated endocytosis) reduces the endocytosis of ∼30–35 % [21]. While the reduction in STx endocytosis is the STxB by 40–50 % but inhibits transferrin endocytosis by likely due to loss of clathrin-mediated internalization, which is ∼70 % [22]. The fact that interfering with clathrin inhibits but also dynamin dependent, the remaining STx endocytosis shows does not abolish STxB endocytosis implies that clathrin- the participation of dynamin- and clathrin-independent independent pathways internalize a proportion of the toxin. endocytic pathways. The expression of dominant negative Further, endocytosis assays performed over longer time frames mutants of RhoA and Cdc42 also partially inhibits STx endo- suggest that clathrin-independent pathways can compensate cytosis, indicating that both RhoA and Cdc42 are required for for the loss of clathrin function with time. Time course analysis optimal STx endocytosis [26]. Like dynamin, Rho GTPases performed in cells treated with anti-clathrin siRNA shows that play a role in clathrin-mediated endocytosis; thus, it is unclear STxB endocytosis is ∼30 % less than control at 10 min but whether the inhibitory effect is due to an effect on clathrin- becomes equal to control by 40 min [23]. Additionally, when dependent or clathrin-independent endocytosis. In contrast, clathrin function is inhibited by K+ depletion or hypertonic dominant negative Cdc42 expression does not affect transferrin treatment (these treatments disperse membrane-associated endocytosis indicating that the requirement of Cdc42 for STx clathrin lattices), there is no difference in STxB internalization endocytosis is